Ultracentrifugal Analysis of the Crystalline Toxin and Isolated Fractions of Clostridium botulinum Type A

Following the chromatographic separation of the crystalline toxin of Clostridium botulinum type A into one fraction (α) containing 5 times the specific activity of the unfractionated preparation and a second fraction (β), only feebly toxic, the native toxin and the two components were analyzed in the ultracentrifuge for homogeneity and Fraction α was analyzed for molecular weight. Under all experimental conditions, crystalline toxin resolved into two peaks with s 20, w values of about 7 and 13, respectively. Fraction β aggregated to a heavier component, with s 20, w of 16 or 23 S, depending upon the pH and ionic strength of the solvent and the concentration of the protein. Fraction α, however, remained homogeneous. The molecular weight of Fraction α, as determined by Yphantis' method, was 128,000 ± 10%. This value is in reasonable agreement with the molecular weight of 150,000 established for this component on Sephadex G-200 columns by Andrews' method. The possibility that Fraction α is a polymer of lower molecular weight units cannot yet be ruled out, nor do we know whether β is the inactive "subunit" proposed by Wagman, an inactive precursor as postulated by Bonventre and Kempe, or a nonspecific contaminant which crystallizes with the toxic α fraction.